Anorexia Nervosa Is Characterized by Increased Adiponectin Plasma ...

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The Journal of Clinical Endocrinology & Metabolism 88(4):1748 –1752 Copyright © 2003 by The Endocrine Society doi: 10.1210/jc.2002-021215

Anorexia Nervosa Is Characterized by Increased Adiponectin Plasma Levels and Reduced Nonoxidative Glucose Metabolism NICOLA PANNACCIULLI, ROBERTO VETTOR, GABRIELLA MILAN, MARNIE GRANZOTTO, ALESSANDRO CATUCCI, GIOVANNI FEDERSPIL, PIERO DE GIACOMO, RICCARDO GIORGINO, GIOVANNI DE PERGOLA

AND

Internal Medicine, Endocrinology, and Metabolic Diseases (N.P., R.G., G.D.P.), Department of Emergency and Organ Transplantation, and Psychiatry (A.C., P.D.G.), Department of Neurological and Psychiatric Sciences, University of Bari, 70124 Bari; and Internal Medicine (R.V., G.M., M.G., G.F.), Department of Medical and Surgical Sciences, University of Padova, 35128 Padova, Italy The aim of the present study was to examine the effects of anorexia nervosa (AN) on adipocytokines (leptin and adiponectin) plasma concentrations and insulin-stimulated glucose disposal in adolescent and young adult women. Adiponectin and leptin plasma levels, along with insulin-stimulated glucose disposal (as measured by the euglycemic-hyperinsulinemic glucose clamp) and oxidative and nonoxidative glucose metabolism (as measured by indirect calorimetry during the last 60 min of the insulin clamp), were measured in 11 anorectic patients and 26 normal-weight healthy female controls. Leptin levels were significantly lower in AN patients, according to the reduced body mass index and their respective fat mass. On the contrary, adi-

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NOREXIA NERVOSA (AN) is an emerging eating disorder affecting mostly adolescent and young adult females in Western societies and characterized by decreased caloric intake, chronically low weight, and resistance to efforts to increase body weight. Patients affected by AN are severely undernourished because of voluntary restriction of food intake lasting several months or years (1). The prolonged starvation causes profound changes in body composition, i.e. a marked loss of lean body mass and adipose tissue. It has been recently demonstrated that adipose tissue serves not only as an energy storage organ but also secretes hormones and metabolites that are thought to regulate insulin sensitivity and energy metabolism (2). Indeed, the term “adipocytokines” was recently coined to describe the adipose-derived bioactive factors modulating the physiological functions of other tissues. Some well known examples among these factors include leptin and adiponectin. Leptin, a 167-amino-acid protein encoded by the obese (ob) gene (3), is expressed almost exclusively in adipose tissue (4), from which it is secreted into the circulation and transported to the hypothalamic area, where it acts as a lipostatic mechanism, modulating satiety and sympathetic nervous systemmediated energy expenditure. Plasma leptin concentrations are strongly related to body fat mass in lean and obese

Abbreviations: AN, Anorexia nervosa; BMI, body mass index; ob, obese gene.

ponectin plasma levels were significantly higher in AN patients than in control women. Likewise, insulin-stimulated glucose disposal and nonoxidative glucose metabolism were significantly lower in AN patients. In conclusion, our study shows that young women affected by AN have higher adiponectin plasma levels than healthy female controls of similar age, despite the presence of an impairment of insulin-stimulated glucose disposal, with a prevalent failure of nonoxidative glucose metabolism. Taken together, these data suggest that the reduction of fat mass may play the major role in the control of adiponectin release, with respect to changes in insulin sensitivity. (J Clin Endocrinol Metab 88: 1748 –1752, 2003)

subjects, increase in the fed state, and decrease rapidly during nutritional deprivation (5, 6). Adiponectin is a 244-amino acid protein with high structural homology to collagen VIII, collagen X, complement C1q (7, 8), and TNF (9). It is produced by the most abundant gene in adipose tissue, the apM1 gene, which is exclusively and abundantly expressed in white adipose tissue. In contrast to all other adipocytokines known to date, plasma adiponectin concentrations were found to be decreased, not increased, in obesity and type 2 diabetes, conditions commonly associated with insulin resistance and hyperinsulinemia (10). The role of adiponectin in later development of type 2 diabetes has been recently assessed in a longitudinal study in the Pima Indian population. Individuals with high concentrations of this protein were less likely to develop type 2 diabetes than those with low concentrations, thus supporting the hypothesis that adiponectin has a key role in the progression from obesity to type 2 diabetes (11). Moreover, it has been shown that, both in obese rats (12) and in humans (13), body weight reduction increased plasma adiponectin despite the reduction of the only tissue of its own synthesis. Interestingly, Havel et al. (14) recently reported a significant increase of plasma adiponectin in normal-weight young adult women, in response to 7 d of energy restriction (⬃600 kcal/ d). On the contrary, depletion of adipose tissue in mice, after treatment with a peroxisome proliferated-activated receptor/retinoid-X receptor inhibitor, resulted in complete absence of adiponectin and in severe insulin resistance, fully

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reversible with supplementation with this adipocytokine (15). On this basis, the profound loss of adipose tissue, caused by prolonged starvation in AN, is likely to be associated with changes in adipocytokines secretion and/or concentration and, consequently, in energy metabolism and insulin-stimulated glucose disposal. Indeed, a significant decrease in plasma leptin levels has been shown to occur in AN (16), thus suggesting that this fall in ob protein circulating concentrations may be involved in the neuroendocrine adaptation to starvation (17). As for adiponectin, to the best of our knowledge, there are no available data concerning the effect of AN on circulating levels of this adipose-specific protein. Therefore, in the current study, we examined the effects of AN on adipocytokines (leptin and adiponectin) plasma concentrations and insulin-stimulated glucose disposal (as measured by the euglycemic-hyperinsulinemic glucose clamp technique) in adolescent and young adult women. Subjects and Methods Eleven women with AN were recruited from the Outpatient Clinic for eating disorders of the University of Bari. Female patients of at least 16 yr of age who met the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, criteria (18) for AN and who had been weightstable for at lease 3 months before the study (as assessed by clinical report) were eligible for the study. All of them provided informed consent (including parental consent for minors). Twenty-six normalweight, healthy women [body mass index (BMI), 18.5–24.9 kg/m2] were recruited, as controls, from the staff and the students of the Section of Internal Medicine, Endocrinology, and Metabolic Diseases of the Department of Emergency and Organ Transplantation of the University of Bari. None of them had any history of eating disorders or other psychiatric illness. Neither control subjects nor anorectic patients had impaired glucose regulation or diabetes mellitus; family history of type 2 diabetes or thyroid, kidney, or liver disease; or were taking any medications known to affect glucose or fat metabolism or the nutritional state. The mean age of the anorectics was 20.6 ⫾ 5.8 yr and was not significantly different from the mean age of 24.6 ⫾ 6.2 yr of the healthy female controls (P ⫽ 0.076). All subjects were measured, in the fasting state, early in the morning, on d 5–7 of the menstrual cycle in control subjects; all the AN patients were amenorrheic. The study protocol was reviewed and approved by the ethics committee of the University of Bari School of Medicine and was performed in accordance with the guidelines proposed in the Declaration of Helsinki. Height and weight were determined according to the standard procedure (19). Height was measured, to the nearest 0.1 cm, with a wallmounted stadiometer. Weight was recorded to the nearest 0.01 kg by using a calibrated computerized digital balance; each subject was barefoot and wore a light bathing suit. Plasma leptin was measured by a specific RIA, which has been previously described in detail (6, 20). Sensitivity was 0.03 ng/liter, and the intra- and interassay coefficients of variation were 0.8% and 8.5%, respectively. Plasma adiponectin was measured by a specific RIA obtained from

Linco Research, Inc. (St. Charles, MO), with minor modifications. Recombinant human adiponectin was used as standard, and a multispecies adiponectin rabbit antiserum was used. The assay buffer contained 10.0 mmol phosphate buffer, pH 7.6, sodium azide (0.09%) and BSA (0.15%). The intra- and interassay coefficients of variation were 3.3% and 8.4%, respectively. Plasma glucose was determined by the glucose-oxidase method (Sclavo, Siena, Italy). Insulin action was assessed, at physiological insulin concentrations, during a hyperinsulinemic-euglycemic glucose clamp, as previously described (21). In brief, after an overnight fast, a primed continuous iv insulin infusion was administered at a constant rate of 40 mU/m2 body surface area䡠min, leading to steady-state plasma insulin concentrations. Plasma glucose concentration was maintained at approximately 5.5 mm, with a variable infusion of a 50% glucose solution. The rate of total insulin-stimulated glucose disposal was calculated for the last 40 min of insulin infusion. Indirect calorimetry (Vmax 29N System; SensorMedics Corporation, Yorba Linda, CA) was used, during the last 60 min of the insulin clamp, to estimate the net rate of carbohydrate oxidation, as previously described (22). Statistical analysis was performed using the STATISTICA 6.0 for Windows (StatSoft Inc., Tulsa, OK) software. The data were first tested for normal distribution by using the Shapiro-Wilk’s test for normality. Leptin and adiponectin concentrations were not normally distributed, so a Kruskal-Wallis test was used to compare the above parameters between AN patients and the control group. As for the other variables, whose distribution was normal, Student’s t test for independent samples was used to evaluate the differences between the AN and control groups. Results are presented as mean and sd for all parameters. A level of significance of P ⬍ 0.05 was used for all data analyses.

Results

General, anthropometric, biochemical, and metabolic data of study subjects are given in Table 1. Adiponectin plasma levels (Fig. 1) were significantly higher in AN patients than in control women (30.5 ⫾ 15.8 vs. 22.6 ⫾ 10.9 ␮g/ml, P ⬍ 0.05). On the contrary, leptin plasma levels (Fig. 1) were significantly lower in AN patients, compared with normalweight controls (2.29 ⫾ 1.55 vs. 8.55 ⫾ 5.44 ␮g/liter, P ⬍ 0.0001), according to the lower BMI (P ⬍ 0.001). Similarly, insulin-stimulated glucose disposal (0.36 ⫾ 0.11 vs. 0.48 ⫾ 0.09 mmol/kg body weight䡠min, P ⬍ 0.01) and nonoxidative glucose metabolism (0.18 ⫾ 0.03 vs. 0.30 ⫾ 0.05 mmol/kg body weight䡠min, P ⬍ 0.001) were significantly lower in AN patients, compared with control women (Fig. 2). Finally, systolic blood pressure was significantly lower in AN patients than in controls (P ⬍ 0.001). No difference between the groups was found for the other variables investigated; in particular, oxidative glucose metabolism was not statistically different between AN patients and control subjects (Fig. 2). Discussion

Adiponectin has emerged most recently as an important adipocytokine that has been clearly demonstrated to have a

TABLE 1. Adiponectin plasma levels and other characteristics of study subjects

Age (yr) BMI (kg/m2) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Fasting glucose (mM) n.s., Not significant.

AN (n ⫽ 11)

C (n ⫽ 26)

P

20.6 ⫾ 5.8 16.4 ⫾ 2.6 92.5 ⫾ 8.2 62.0 ⫾ 10.1 3.92 ⫾ 0.59

24.6 ⫾ 6.2 20.7 ⫾ 2.8 106.7 ⫾ 11.7 67.7 ⫾ 7.4 4.01 ⫾ 0.49

n.s. ⬍0.001 ⬍0.001 n.s. n.s.

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FIG. 1. Adiponectin and leptin plasma levels in anorectic patients (AN) and in normal-weight healthy female controls (C).

variety of functions, including antiatherogenic, antiinflammatory, and insulin-sensitizing properties (23–25). The most original and striking result of the present study is that anorectic patients had higher adiponectin plasma levels, compared with normal-weight healthy female controls, despite that insulin sensitivity was lower in the AN group than in controls. This finding is quite surprising if it is taken into account that the closest correlate of plasma adiponectin has been shown to be the degree of insulin resistance (10); moreover, it has been demonstrated that administration of adiponectin increases insulin sensitivity in animal models (15). We do not have a clear-cut explanation for our apparently paradoxical findings; however, several hypotheses may be formulated. Adiponectin has been shown to be suppressed in states of insulin resistance, such as type 2 diabetes and obesity (8, 10); however, it is largely unknown which factors might contribute to this down-regulation. Lower plasma adiponectin levels in obesity, despite that adipose tissue is the only source of this protein, suggest a negative feedback on adiponectin production. In this respect, several experimental evidences [including the demonstration that expression of adipogenic genes, in general, and adiponectin, in particular, from adipose tissue of animals and humans is suppressed or downregulated in obesity and type 2 diabetes (8, 26)] argue for the existence of such a feedback inhibitory pathway. Therefore, body weight reduction would result in disinhibition and, consequently, elevation of adiponectin concentrations. This negative feedback pathway between adipose tissue and adiponectin expression might explain higher plasma

FIG. 2. Insulin-stimulated glucose disposal, nonoxidative glucose metabolism, and oxidative glucose metabolism in anorectic patients (AN) and in normal-weight healthy female controls (C).

adiponectin levels in AN patients, as compared with agematched, normal-weight female controls, that we found in the present study. Indeed, Havel et al. (14) recently reported a significant increase of plasma adiponectin in young adult women in response to 7 d of energy restriction (⬃600 kcal/d); this study is of outstanding interest for us because the adiponectinincreasing effect of caloric restriction has been demonstrated in a group of normal-weight women, thus suggesting that weight loss is able to raise circulating adiponectin concentrations even in the absence of overweight or obesity. Moreover, we cannot exclude a role of reduced leptin concentrations in determining the elevation of adiponectin plasma levels. Indeed, it is well known that there are relevant interactions between leptin and other cytokines; these interactions could result in an additive, synergistic, or even an-


tagonistic effect, thus suggesting the existence of positive and negative feedback systems dependent on cytokine-cytokine interactions (27, 28). Finally, it can be hypothesized that elevated circulating adiponectin concentrations might represent a compensatory mechanism for the reduced insulin-stimulated glucose metabolism in AN patients, compared with normal-weight controls. Previous studies regarding insulin sensitivity in anorectic patients gave conflicting results. In particular, insulin-stimulated glucose disposal has been reported to be normal (29), enhanced (30, 31), or decreased (32) in AN subjects. In our study, we found a reduced insulin-stimulated glucose disposal in the AN group, with prevalent impairment of nonoxidative glucose metabolism and no difference in glucose oxidation, thus suggesting that insulin stimulates glucose oxidation more than glucose storage in AN patients. Nonoxidative glucose metabolism represents more than 90% storage of glucose as glycogen in muscle and, interestingly, lower glycogen concentrations have been reported in skeletal muscle of anorectic patients (33). Therefore, our results indicate that a nonoxidative pathway of glucose metabolism is resistant to the action of insulin in AN; a similar situation is commonly observed during prolonged starvation, i.e. a further condition characterized by lower sensitivity to insulin. Insulin resistance in anorectic patients may well be a compensatory response to energy deprivation; in fact, glucose metabolism is directed toward immediate oxidation more than storage. The conflicting results of the studies addressing the issue of insulin sensitivity in AN may be attributable to differences in the techniques for evaluating insulin action; in particular, our finding of reduced insulin-stimulated glucose disposal in anorectic patients, as compared with normal-weight controls, is consistent with the only study in which insulin sensitivity was measured by the glucose clamp technique (32). We cannot indicate, with certainty, what factor is responsible for the uncoupling between adiponectin plasma levels and insulin-mediated glucose metabolism; in this respect, we can hypothesize that some cytokines, such as TNF-␣, may play an important role. Indeed, it has been demonstrated that plasma concentrations of TNF-␣, referred to as a potential mediator of insulin resistance (34), are increased in anorectic patients independently of body fat, thus suggesting that the adipose tissue may not be the immediate source of this cytokine in AN (35). Overall, the above metabolic perturbation of anorectic patients may be interpreted, from a finalistic point of view, as a defensive mechanism directed to prevent such undernourished patients from hypoglycemia. We are aware of the fact that a weak point of this study is the relevant difference of age, even though not statistically significant, between AN patients and normal-weight controls. However, we do not believe that this difference played a crucial role in relation to adiponectin and leptin concentrations, because there have been several studies that could not find a significant relationship of age with both adiponectin (36) and leptin (37) circulating levels. This study also supports previous reports that anorectics have significantly lower levels of plasma leptin than normal (16, 38), thus suggesting that this cytokine is not involved in the reduction of food intake associated with AN, and that

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shifts of energy balance (either positive, i.e. body weight gain, or negative, i.e. body weight loss) are accompanied by changes in circulating leptin levels reflecting total body fat mass. Furthermore, it has been demonstrated that leptin concentrations decrease during nutritional deprivation more rapidly than would be expected on the basis of weight loss (39). In conclusion, our study demonstrates that young women suffering from AN have higher adiponectin plasma levels and lower insulin-stimulated glucose disposal, with prevalent impairment of nonoxidative glucose metabolism, than normal-weight healthy female controls of similar age. The increase in adiponectin concentrations might be thought of as a compensatory mechanism for insulin resistance of AN patients, but further studies are needed to confirm this possibility. Acknowledgments The excellent technical assistance of Marilena Tormene and Sonia Leandri is greatly appreciated. Received August 1, 2002. Accepted January 2, 2003. Address all correspondence and requests for reprints to: Giovanni De Pergola, M.D., via Putignani 236, 70122 Bari, Italy. E-mail: g.depergola@ endo.uniba.it. This work was supported by a grant from the Italian Ministry of University and Research (to G.F.).

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